Investigating DDX3X as a sex-specific translational regulator associated with intellectual disability

项目摘要 我们对DDX 3X（一种高危基因）的遗传学和神经生物学的理解存在根本性的差距 X连锁智力残疾（ID）占女性不明原因诊断的2%。DDX3X 突变显示了一个令人困惑的性别二分法：大多数突变是从头开始的，只在女性中发现;少数突变是在女性中发现的。 男性的突变遗传自健康的母亲。DDX 3X调节mRNA翻译，但 神经元中的作用机制、靶基因和临床突变的影响尚未研究。 此外，性别的影响仍然未知。迫切需要填补这些空白，因为在我们这样做之前， 实施使用DDX 3X作为治疗靶点的策略仍然遥不可及。长期目标是 绘制ID背后的突触机制并确定新的治疗靶点。总体目标是 捕获由DDX 3X突变引起的性别特异性突触变化。核心假设是， DDX 3X以性别特异性方式调节突触翻译和突触发生：突变影响这些 在男性和女性的过程不同，导致性别偏见的患病率和严重程度的条件。 基本原理是，一旦我们知道DDX 3X如何调节突触翻译以及性别如何影响它， 可以开发基于机制的精确治疗。中心假设将通过以下方式进行检验： 两个特定目的：1）分析DDX 3X在突触翻译和突触发生中的作用;以及，2）确定 性别特异性DDX 3X突变的影响。在目标1下，将应用生物化学和结构方法 研究DDX 3X介导的小鼠突触翻译。基于病毒的核糖体亲和力翻译 纯化（vTRAP-seq）将用于绘制雄性和雌性小鼠中DDX 3X调控的mRNA图谱 突触一种新的DDX 3X小鼠的雄性和雌性单胚胎神经元中的树突棘分析 模型将用于评估在突触发生中的作用。在目标2下， 突变将在结构上和生物化学上建模。这些突变将被引入培养的神经元中 和脊柱表型比较突变和性别，以了解为什么女性致病 突变更严重。所有这些方法都是申请人专业知识的一部分。这一建议具有创新性 因为它解决了一个新的ID高风险基因的生物学，根据一个核心但被忽视的方面： 性它是创新的，因为它模拟了神经元中的患者特异性突变。它也是创新的，因为它 整合了遗传学、结构生物学、生物化学、分子生物学和神经科学， 小鼠模型和新型DDX 3X小分子抑制剂。这个应用程序是重要的，因为它将至关重要 进一步加深我们对DDX 3X综合征和ID的理解。结果将揭示基本的 突触发生的各个方面，从而推进健康和疾病中大脑发育的知识。的 这些结果有望产生积极的影响，因为它们将精确地确定新的分子靶点 这将为临床遗传学护理提供信息，并为家庭带来短期和长期的好处。

PROJECT SUMMARY There are fundamental gaps in our understanding of the genetics and neurobiology of DDX3X, a high-risk gene for X-linked intellectual disability (ID) accounting for up to 2% of unexplained diagnoses in females. DDX3X mutations display a puzzling sexual dichotomy: most mutations are de novo and found only in females; the few mutations in males are inherited from healthy mothers. DDX3X regulates mRNA translation, but the mechanisms of action in neurons, the target genes, and the impact of clinical mutations have not been studied. Also, the influence of sex remains unknown. There is a critical need to fill these gaps because, until we do so, implementing strategies that use DDX3X as a therapeutic target remains out of reach. The long-term goal is to map the synaptic mechanisms underlying ID and identify new therapeutic targets. The overall objective is to capture the sex-specific synaptic changes resulting from mutations in DDX3X. The central hypothesis is that DDX3X regulates synaptic translation and synaptogenesis in a sex-specific manner: mutations impact these processes differently in males and females, leading to sex biases in prevalence and severity of the condition. The rationale is that, once we know how DDX3X regulates synaptic translation and how sex influences it, mechanism-based precision therapeutics can be developed. The central hypothesis will be tested by pursuing two Specific Aims: 1) Analyze the role of DDX3X in synaptic translation and synaptogenesis; and, 2) Determine the effects of sex-specific DDX3X mutations. Under Aim 1, biochemical and structural methods will be applied to study DDX3X-mediated translation in mouse synapses. Viral-based Translating Ribosome Affinity Purification (vTRAP-seq) will be used to map the mRNAs regulated by DDX3X in male and female mouse synapses. Dendritic spines analyses in male and female single-embryo neurons from a novel DDX3X mouse model will be used to assess the role in synaptogenesis. Under Aim 2, two male- and three female-pathogenic mutations will be structurally and biochemically modeled. The mutations will be introduced in cultured neurons and the spine phenotype compared across mutations and sexes to understand why female-pathogenic mutations are more severe. All these methods are part of the applicant's expertise. This proposal is innovative because it addresses the biology of a novel ID high-risk gene in light of a central and yet overlooked aspect: sex. It is innovative because it models patient-specific mutations in neurons. It is also innovative because it integrates genetics, structural biology, biochemistry, molecular biology and neuroscience, and uses a novel mouse model and novel DDX3X small-molecule inhibitors. This application is significant because it will critically advance our understanding of DDX3X syndrome and ID more broadly. The results will expose fundamental aspects of synaptogenesis, thus advancing the knowledge of brain development in health and disease. The results are expected to have a positive impact because they will pinpoint novel molecular targets for precision medicine, while informing clinical genetics care, with both short-term and long-term benefits for families.